Ubiquitin Ligase TRIM39 Control of Zika Virus Infection - Summary Viruses require a variety of host cell proteins to enable them to replicate, and identification of host factors that contribute to virus replication, fitness, and tropism is a top priority for basic and applied research. Zika virus (ZIKV) is a Flavivirus known to cause significant human disease and mortality and can cross the placental barrier to infect unborn children. Despite its direct impact on human health there are currently limited therapeutic options available. It is essential therefore to identify the factors that contribute to ZIKV infection and understand their modes of action. Cellular ubiquitin (Ub) systems are important for regulating fundamental eukaryotic processes and mediating both innate and adaptive immunity. Ub pathways are often appropriated by viruses to directly or indirectly influence their replication. We used an original live cell-based high-throughput screen was used to identify Ub-related genes that are required for ZIKV replication. Rigorous primary, secondary, and counter screening was used to identify host factors that impact ZIKV replication. Based on the intensity and reproducibility of its impact on ZIKV replication, TRIM39, a Ub ligase not previously connected to ZIKV, was advanced as a promising target for further investigation. Additional results demonstrate that TRIM39 disruption restricts both 1947 and 2015 ZIKV isolates, decreases infectious virus production by ³100-fold, limits ZIKV RNA and protein production, and reduces antiviral responses. These properties suggest that TRIM39 and its Ub substrates have high potential for development as anti-ZIKV therapeutic targets. However, little is known about TRIM39 itself or how it may interface with ZIKV. Experiments indicate TRIM39 level is decreased during ZIKV infection, and that it is specifically antagonized by the ZIKV NS5 protein, a multifunctional viral protein responsible for RNA synthesis and innate immune evasion, leading to proteasomal degradation. We find that NS5 engages the TRIM39 SPRY domain in a complex that leads to this viral hijack, suppression, or antagonism. As a previously unrecognized host protein that can modulate ZIKV infection, we postulate that TRIM39 is needed in the positive progression of ZIKV infection and may target inhibitory or antiviral factors that otherwise would hinder ZIKV replication. The ZIKV NS5 protein modulates TRIM39 actions via a protein interaction and degradation. Three Aims will test these hypotheses, reinforce TRIM39’s significance for ZIKV and other RNA viruses, and reveal its roles in ZIKV infections. We will (i) expand analysis of TRIM39 for ZIKV in placental trophoblast derived host cells, and for both closely related (Dengue) and unrelated (IAV, VSV) RNA virus infections, (ii) identify TRIM39 interaction partners and Ub substrates in uninfected and infected cells, and (iii) determine the mechanistic basis of NS5-TRIM39 interaction and its impact on the ZIKV life cycle. These goals will produce a more complete understanding of TRIM39 in ZIKV biology, reveal the mechanisms underlying its essential role in infections, and authenticate a new mediator of virus infection.
